Past studies have established that hyperlipidemic atherosclerosis arises as a consequence of the deposition and retention of serum lipoproteins in the arterial wall. Although macrophage-foam cell formation has been extensively studied, little is known about how vascular smooth muscle cells (VSMCs) become foam cells. Internalization of matrix retained and aggregated LDL may involve the actin cytoskeleton in manner that distinguishes this process from the endocytosis of soluble LDL. The strategy of this proposal is to define novel mechanisms of LDL uptake through the modulation of the actin cytoskeleton to identify molecular target for the control of foam cell formation in VSMCs. The critical observation that forms the central hypothesis of this application is that under patho-physiological conditions, nucleotide release from blood-derived and vascular cells (e.g., aggregating platelets, leukocytes, endothelial and smooth muscle cells) activates SMC P2Y2 nucleotide receptors (P2Y2Rs) leading to the rearrangement of the actin cytoskeleton and cell motility. We hypothesize that the P2Y2R may regulate foam cell formation in VSMCs thereby contributing to the formation of advanced atherosclerotic lesions. To test this hypothesis, we have obtained VSMCs from wild and the P2Y2R-/~ mice for evaluating the role of this receptor in the internalization of matrix-bound LDL in VSMCs through the following specific aims: Specific Aim i: To determine the role of P2Y2R-mediated cytoskeleton rearrangement in the uptake of matrix-hound aggregated LDL. Study i: To determine the effect of P2Y2R agonists in the uptake of matrix-bound aggregated LDL in cultured VSMCs obtained from wild type and P2Y2R-/- mice using confocal and electron microscopy. Study 2 will use cultured SMCs from P2Y2R /- mice transduced with adenoviruses encoding a mutant P2Y2R defective in FLN-A binding to determine the effect of the loss of P2Y2R/FLN-A interactions in the internalization of modified LDL. Study 3 will use inhibitors and dominant negative mutants of RhoA, Rac and cd42 to determine whether small GTPases are critical for P2Y2R mediated LDL uptake. Specific Aim 2; To determine the mechanisms whereby P2Y2R agonists modulate LRP expression in SMCs. Study 4 will use real-time PCR to determine the effects of P2Y2R agonists in LRP mRNA expression in SMCs isolated from wild type and P2Y2R-/- mice . Study 5 will determine whether LRPi is regulated at the transcriptional level and if there is a regulatory element in the LRP promoter that is responsive to P2Y2R agonists. Public Health Relevance: Accumulation of cholesterol is a hallmark of cardiovascular disease the main cause of death in the USA. This proposal seeks to identify molecular mechanisms that may lead to the prevention of lipid accumulation in the arteries, the main cause of atherosclerosis.